This project focuses on the biochemical mechanisms involved in the formation and maintenance of ionic transport gradients and their dissipation via conductive pathways in excitable tissues. Single channel current fluctuations from Ca2+ release channels in isolated membrane vesicles of sarcoplasmic reticulum prepared from skeletal muscle were recorded using the patch clamp technique. The level of Ca2+ conductance and pattern of sensitivity to activators and inhibitors of Ca2+ release support participation of this channel in the mechanism of activation of muscle contraction. Measurements of ATP-dependent Ca2+ uptake, Ca2+ ATPase function and passive Ca2+ diffusion support the view that reduced active Ca2+ uptake rates in cardiac sarcoplasmic reticulum from old rats may be due to an increased Ca2+ efflux rate mediated by a Ca2+-activated Ca2+ channel. Investigation of the transient phase of Ca2+ uptake by the Na+/Ca2+ exchanger in cardiac sarcolemmal vesicles revealed the presence of a biphasic burst phase that was inhibited by intravesicular Na+. The results are consistent with a mechanism in which Na+ stabilizes the formation of a Na+-specific binding conformation that undergoes slow conversion to a Ca2+-specific form upon addition of Ca2+. A transient burst phenomenon was also observed in the time course of Na+ uptake mediated by the Na+/H+ exchanger in kidney brush border membranes. The pH dependence of the burst favors a consecutive as opposed to a concerted movement of Na+ and H+ during the first turnover of the system. Solubilization of Na+,K+-ATP-ase was shown to activate turnover of the phosphoenzyme in fashion similar to that produced by K+. Disruption of intersubunit contacts and exposure of the catalytic site to H2O could account for this behavior. Stopper-flow fluorescent studies of lactose accumulation in E. coli membrane vesicles have demonstrated complex behavior indicative of sequestration followed by partitioning of the substrate into the inner leaflet of the membrane.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Intramural Research (Z01)
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